Information Transfer:
DNA replication, transcription and translation.

In very general terms, what does a chromosome contain?

• Information, genetic information to carry out the characteristics of life -- precise self replication, ability to exchange energy with the environment, etc.

• Information storage
  • DNA replication
• Information transfer
  • DNA transcribed into RNA
  • DNA's function in information transfer

• Central Dogma:
  DNA-->RNA-->unfolded protein-->native, folded protein

• replication, DNA --> DNA
• transcription, DNA --> RNA
• translation, RNA --> unfolded protein --> folded protein

• Translation of linear information, a sequence of nucleotides, into 3-D information, the structure of a protein.

Principle of Information Transfer.
The maintenance of the high degree of structural and functional organization in an organism is an essential prerequisite for the continuation of life. The perpetuation of life depends on sequential and conformational molecular information.
• Concept 1: The information contained in the linear sequence of monomers in a polymer determines higher order structure and function.
• Concept 2: The flow of biochemical information is from nucleic acid to protein, generally DNA to RNA to Protein.
• Concept 3: Information flows from molecules in which sequencial information predominates (nucleic acids) to molecules in which conformational information predominates (proteins).
Central Dogma: DNA --> RNA --> unfolded protein --> folded protein

What are the differences between DNA and RNA?

• base composition: RNA = AGCU, DNA = AGCT
• carbohydrate: RNA = ribose, DNA = deoxyribose
• structure: RNA = single stranded, DNA = double helix

RNA
• usually single stranded
• linear polymer of ribonucleotides.
• Some secondary and tertiary structure but often ill-defined.

• messenger RNA = mRNA, information transfer
• transfer RNA = tRNA, information transfer
• ribosomal RNA = rRNA, structural
• small nuclear RNA = snRNA, ribozymes, RNA processing.

Information Transfer:
DNA replication, transcription and translation.

What is replication?
Transfer of genetic information from one generation to the next.
DNA-directed DNA synthesis: replication of the genome.

What is the structural basis for the precise duplication of the genome?

• The Watson-Crick structure of DNA: the strands are complementary, the nucleotide sequence in one automatically specifies the other.
• The enzyme, DNA polymerase III, is very accurate: it has proof reading capabilities.

Is replication conservative or semi-conservative?
What does that mean?

• Is the parental genome of double stranded DNA fully conserved in the parental cell or is it split equally (semi-conserved) between two daughter cells?

Replication is semi-conservative.
What is the evidence for semi-conservative replication?

Classical experiments of Meselson and Stahl. Label DNA with *heavy isotope* N15 and allow replication in light N14: distinguish heavy, light and hybrid DNA by centrifugation.
Results: after 1 generation, each genome contains a hybrid N15-N14 DNA; after 2 generations, there are 2 hybrid and 2 light (N14-N14) genomes.

• Each strand of DNA serves as a template for the synthesis of its complement.
• The strands separate and each is used as a template for the synthesis of a daughter strand.
• The two new double helices each contain half the parental DNA.
• This process produces a replication fork

Is replication uni-directional or bi-directional?

• Bi-directional
  
• Two replication forks proceeding from the origin.

What is the major replication enzyme?
DNA polymerase III, a DNA-directed DNA polymerase

• Synthesis is 5'-->3'
• Substrates are deoxynucleoside triphosphates (to make deoxyribonucleic acid)
• Proof reading , errors removed by 3'--5' exonuclease
• Processivity is very high (the ability of the enzyme to replicate a large tract of DNA before *falling* off)
• Replication requires DNA unwinding by enzymes termed helicases: these enzymes unwind the DNA helix before the replication fork and wind it up again afterwards.

DNA damage by UV radiation or chemicals is repaired by other DNA polymerases.
UV-damage results in adjacent T residues in one strand becoming covaletly linked to each other, producing a thymine dimer. This causes the double helix to become distorted -- kinky.
Xeroderma pigmentosa is a genetic disorder in which patients cannot carry out UV-radiation repair. They are very prone to skin cancer from an early age.

What is Transcription?

• Copying a gene as RNA
• DNA-directed RNA synthesis from a gene

What is a gene?

• There is no good definition of a gene!
• A sequence of DNA that is transcribed from specific start to specific stop base sequences.
• Beadle and Tatum, working with the eukaryote mold Neurospora crassa, concluded that one gene codes for one protein.
• But what about genes that code for RNA's like rRNA and tRNA?
• A gene is a sequence of DNA that is transcribed into a single RNA as defined by specific start and stop sequences of bases.
• Note the circularity of the argument!
• But the single RNA may be polycistronic!

What does that mean?

• A cistron is synonymous with a gene.
• A polycistronic RNA results from the transcription of an operon.

What's an operon?

• A genetic unit containing several genes with related functions: the bacterial operon for lactose (milk sugar) metabolism contains 3 genes coding for 3 different proteins.
• An operon is transcribed as a single unit, a polycistronic messenger RNA (mRNA) that codes for more than one gene product.

Name 4 types of RNA.
What are their functions?

• mRNA, messenger RNA that is translated into protein
• rRNA, ribosomal RNA that, together with ribosomal proteins, forms a structural scaffold for the translation of mRNA, the ribosome
• tRNA, transfer RNA, a specific carrier of amino acids
• snRNA, small nuclear RNA involved in processing of mRNA in the nucleus

What is the major transcription enzyme?
RNA polymerase, a DNA-directed RNA polymerase

• RNA synthesis is 5'-->3'
• substrates are ribonucleoside triphosphates ( to make ribonucleic acid)
• begins at the promoter, 5' end of the gene
• processivity is very high, proceeds to 3' end of gene without stopping or falling off the gene
• proof reading by precise Watson-Crick base pairing, A=U and G=C

• Transcription of some genes is constitutive = housekeeping genes
• Transcription of other genes is in response to a stimulus = inducible genes

• exons are coding regions, and
• introns are non-coding regions of the mRNA transcript
• exons and introns are found in most, but not all, eukaryote genes
• introns have to be spliced out before the mRNA is translated
• splicing is by snRNA's acting as enzymes, or ribozymes, an example of the catalytic function of RNA

Translation

• Synthesis of a linear polymer of amino acids from a linear polymer of nucleotides

Where does it occur?
On the ribosome, a rRNA-protein complex that provides:

• a scaffold for mRNA
• sites for the docking of tRNA charged with a specific amino acid
• an enzyme for peptide bond synthesis between amino acids
• an enzyme for translocation of the mRNA through the ribosome

What is the function of tRNA?

• Carrier of a specific amino acid during translation

What is the structure of tRNA?

• secondary structure has some base-pairing --> cloverleaf
• information transfer at the anti-codon loop, complementary to the codon
• note the importance of H-bonds in the genetic code
• tertiary structure is L-shaped which places the amino acid far from the codon-anticodon site
• degeneracy of the code produces wobble

What is the genetic code?
A sequence of 3 nucleotides forms a codon

• unambiguous, each codon specifies an amino acid, or start, or stop
• degenerate, some amino acids have multiple codons
• 2-letters often sufficient, specifiy hydrophobic and hydrophillic amino acids

What is the enzyme that charges tRNA with an amino acid?
an aminoacyl-tRNA synthetase

• it has proof reading capabilities through the precise fit of amino acid and tRNA
• energy provided by ATP: energy for the formation of aminoacyl-tRNA and for proof reading
• there are at least 20 synthetases, isoaccepting for the tRNA's coding for a single amino acid

What is the mechanism of translation?

• mRNA forms a large complex with the ribosome and protein factors
• together they guide in the correct aminoacyl-tRNA
• correct amino acid specified by codon-anticodon base pairing (H-bonds)
• protein factors have proof reading capability--energy provided by GTP
• an enzyme catalyzes polymerization of two amino acids, peptide (amide)bond formation between two amino acids (see Chemistry lecture)
• an enzyme catalyzes movement of mRNA through the polymerization site: energy provided by GTP
• mRNA translated from 5'--> 3', same direction as it is synthesized

Reprise:

• Flow of information: central dogma
• DNA--> RNA-->linear amino acid sequence --> 3D-conformation of protein

But some viruses have only RNA as their genome: no DNA.

How do they carry out information transfer?

How do they get around the unidirectional flow of information in the central dogma?

• Use an enzyme called reverse transcriptase to transcribe RNA into DNA.
• Example: HIV, a retrovirus
• Then, use central dogma.
• For HIV:

  RNA-->DNA--> mRNA --> linear amino acid sequence --> 3D-conformation of protein

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Bich 107 lecture notes on Information Transfer were last updated 09/23/03

Comments to Martyn Gunn